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Minimum inhibitory concentration,Antibiotic Sensitivity Testing

Minimum inhibitory concentration,Antibiotic Sensitivity Testing

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Minimum inhibitory concentration Presentation Transcript

  • 1. Dr.T.V.Rao MD
    Antibiotic Sensitivity testing Minimum inhibitory concentrationskill based learning
    Dr.T.V.Rao MD
    1
  • 2. The responsibility of the microbiology laboratory includes not only microbial detection and isolation but also the determination of microbial susceptibility to antimicrobial agents. Many bacteria, in particular, have unpredictable susceptibilities to antimicrobial agents, and their susceptibilities can be measured in vitro to help guide the selection of the most appropriate antimicrobial agent.
    Why antibiotic susceptibility testing
    Dr.T.V.Rao MD
    2
  • 3. What is susceptibility to Antibiotics
    The term susceptible means that the microorganism is inhibited by a concentration of antimicrobial agent that can be attained in blood with the normally recommended dose of the antimicrobial agent and implies that an infection caused by this microorganism may be appropriately treated with the antimicrobial agent. The term resistant indicates that the microorganism is resistant to concentrations of the antimicrobial agent that can be attained with normal doses and implies that an infection caused by this microorganism could not be successfully treated with this antimicrobial agent.
    Dr.T.V.Rao MD
    3
  • 4. Antimicrobial resistance results from ….
    Results from misuse, overuse, under/ inadequate use of
    antimicrobials
    Costs money, lives and undermines effectiveness of health delivery programs
    Threat to global stability and national security
    WHO Global Strategy for Containment of Antimicrobial
    Resistance:
    Intervention framework to slow emergence and reduce the spread of antimicrobial resistant microorganisms
    Dr.T.V.Rao MD
    4
  • 5. Antibiotic resistant infections
  • 6. Antimicrobial susceptibility tests
    Minimum inhibitory concentration [MIC]
    The smallest concentration of antibiotic that inhibits the growth of organism
    Liquid media (dilution) allows MIC estimation
    Solid media (diffusion)
    Disk diffusion (Kirby-Bauer)
    E-tests
    Allows MIC estimation
    Beta lactamase production: quick screening method
    Dr.T.V.Rao MD
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  • 7. Dr.T.V.Rao MD
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  • 8. Susceptibility testing: The big gaps
    MICs help, but hard to standardize
    Correlations appear possible based on individual isolates. Broad correlations based on multiple isolates are still lacking
    Understanding this helps a lot when trying to correlate outcome with MIC
    - Some patients get better despite MICs
    - Some patients just don’t get better despite MICs
    No rule when it correlates/not correlates
    Dr.T.V.Rao MD
    8
  • 9. The tube dilution test is standard method
    The tube dilutiontest is the standard method for determining levels of microbial resistance to an antimicrobial agent Serial dilutions of the test agent are made in a liquid microbial growth medium which is inoculated with a standardized number of organisms and incubated for a prescribed time. The lowest concentration (highest dilution) of test agent preventing appearance of turbidity (growth) is considered to be the minimal / minimum inhibitory concentration (MIC). At this dilution the test agent is bacteriostatic.
    Dr.T.V.Rao MD
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  • 10. The MIC or minimum inhibitory concentration test determines antimicrobial activity of a material against a specific bacteria.
    The most commonly employed methods are the tube dilution method and agar dilution methods. Test products that are not clear or precipitate the growth media are tested by agar dilution methods which is about same as tube dilution method except dilutions are plated on agar
    minimum inhibitory concentrationtest determines
    Dr.T.V.Rao MD
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  • 11. The Minimum Inhibitory Concentration (MIC) is the smallest concentration of an antimicrobial agent that inhibits the growth of bacteria. The value is obtained in a highly mechanized fashion, but this procedure only provides interval censored reading. It is often of interest to use data collected from complex experiments to see how the mean MIC is affected by different factors
    Minimum Inhibitory Concentration (MIC)
    Dr.T.V.Rao MD
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  • 12. The minimal bactericidal concentration (MBC) or the minimum lethal concentration (MLC) of an antibacterial which is defined as the maximum dilution of the product that will kill a test organism can be determined by sub culturing last clear MIC tube onto growth medium and examining for bacterial growth. Serial dilutions are made of the products in bacterial growth media.
    The minimal bactericidal concentration
    Dr.T.V.Rao MD
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  • 13. A minimum bactericidal concentration test
    Figure 10.12
    Dr.T.V.Rao MD
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  • 14. Minimum inhibitory concentration (MIC)Practice the exercise for skills
    1.    Grow cultures up overnight.2.    The following day, inoculate a fresh culture with a 1:10-20 dilution and grow up to OD600 0.4. 3.    Dilute to OD600 0.0005 (1:800) in THB (or relevant media).4.    Dilute this 1:200 and drop about 25µl on THA (or relevant agar plates) to confirm equal starting inoculums.5.    Put 50µl of the 0.0005 cultures in a 96 well plate in triplicates.6.    Add 50µl per well of the substance you want to test (ie. antimicrobial peptide, antibiotic) to each well, keeping in mind the final concentration will be half the original concentration you are adding. It is good to test 1:2 dilutions (in THB, or relevant media) of this substance.7.    Incubate overnight and check by eye or OD600 24 hours later to determine what concentration inhibited the growth of the different strains of bacteria.
    Dr.T.V.Rao MD
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  • 15. Dilution in liquid broth
    MIC
    Tubes containing increasing antibiotic concentrations
    Incubation during 18 hr at 37°C
    Tedious
    Bacterial growth
    Inhibition
    0 (Control) 0,25 0,50 1 2 4 8 mg/l
    Dr.T.V.Rao MD
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  • 16. Dr.T.V.Rao MD
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  • 17. Dr.T.V.Rao MD
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  • 18. Standard strains for quality assurance
    Precision and accuracy ensured through control strains
    Known susceptibility to antimicrobial agents
    Standard strains include
    Staphylococcus aureus ATCC 25923
    Escherichia coli ATCC 25922
    Pseudomonas aeruginosa ATCC 27853
    Dr.T.V.Rao MD
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  • 19. Use standardized reference
    National Committee for Clinical Laboratory Standards (USA)
    Other norms
    Canadian
    Chinese
    National
    Do not confuse the different tables
    Choose one for everything
    Different standardsCLSI Standards followed all over the world
    Dr.T.V.Rao MD
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  • 20. Culture media: Muller-Hinton
    Reagents: disks
    Size of the inoculums
    Incubation condition
    Control with reference strains
    Reading inhibition diameters (accurate measurement)
    Knowledge of staff
    Critical points in quality assurance- Organise your Laboratory with
    Dr.T.V.Rao MD
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  • 21. Principles of Minimum inhibitory concentration
    In minimum inhibitory concentration dilute on a log2 scale each antimicrobial agent in broth to provide a range of concentrations and to inoculate each tube or, if a micro plate is used, each well containing the antimicrobial agent in broth with a standardized suspension of the microorganism to be tested. The lowest concentration of antimicrobial agent that inhibits the growth of the microorganism is the minimal inhibitory concentration (MIC). The MIC and the zone diameter of inhibition are inversely correlated . In other words, the more susceptible the microorganism is to the antimicrobial agent, the lower the MIC and the larger the zone of inhibition. Conversely, the more resistant the microorganism, the higher the MIC and the smaller the zone of inhibition.
    Dr.T.V.Rao MD
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  • 22. Minimum Inhibitory Concentration (MIC) :
    Principle:
    • The tube dilution test is the standard method for determining levels of resistance to an antibiotic.
    • 23. Serial dilutions of the antibiotic are made in a liquid medium which is inoculated with a standardized number of organisms and incubated for a prescribed time.
    • 24. The lowest concentration of antibiotic preventing appearance of turbidity is considered to be the minimal inhibitory concentration (MIC).
    Dr.T.V.Rao MD
    22
  • 25.
    • Different concentrations of Gentamycin in Nutrient broth:
    Conc. in mcg/ml
    0.1 0.2 0.4 0.8 1.6 3.1
    Gentamicin, generally considered a bacteriocidal antibiotic, for this bacterium, has an MIC of 0.8 mcg/ml
    Dr.T.V.Rao MD
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  • 26.
    • Different concentrations of Tetracycline in Nutrient broth:
    Conc. in mcg/ml
    0.1 0.2 0.4 0.8 1.6 3.1 6.3 12.5
    Tetracycline, generally considered a bacteriostatic antibiotic, for this bacterium, has an MIC of 1.6 mcg/ml
    Dr.T.V.Rao MD
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  • 27. Minimum inhibitory concentration test
    Figure 10.10
    Dr.T.V.Rao MD
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  • 28. Interpretation
    The main concept is the “clinical categorisation"
    Strains are sorted according to level of Minimal Inhibitory Concentration (MIC) versus reference breakpoints
    c and C are the minor and major breakpoints
    Dr.T.V.Rao MD
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  • 29. Understanding breakpoints
    Words of laboratory specialists
    It is not possible to work alone
    Breakpoints are the expression of a consensus among the scientific community at a given time in a country
    Breakpoints are determined using two approaches
    Pharmacological concept
    Epidemiological concept
    Dr.T.V.Rao MD
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  • 30. Inherited resistance mechanism
    Wild type
    c
    C
    MIC
    The epidemiological concept for breakpoints
    Dr.T.V.Rao MD
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  • 31. The pharmacological concept for breakpoints
    The concentration range tested for a drug and the
    interpretative criteria for various categories are based on
    extensive studies that correlate with
    Serum achievable levels for each antimicrobial agent
    Particular resistance mechanisms
    Successful therapeutic outcome
    In practice situations the entire range may not be used for
    decision making and therefore the concept of breakpoint
    concentration
    Dr.T.V.Rao MD
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  • 32. From breakpoints to interpretation
    Measuring antimicrobial sensitivity of a strain isolated from a patient, to determine its status as S, I or R is an individual problem
    Defining the status of a bacterial species or genus is an epidemiological problem distributed across time and space that requires monitoring
    Dr.T.V.Rao MD
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  • 33. Sometime the agent can still be used
    Higher doses required to ensure efficacy
    Agent may be efficacious if concentrated in vivo in an infected body fluid (e.g., urine)
    Sometimes there is uncertainty
    Intermediate resistance may represent a “buffer” zone that prevents strains with borderline susceptibility from being incorrectly categorized as resistant
    Interpreting intermediate resistance
    Dr.T.V.Rao MD
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  • 34. Common interpretation problems
    Results depends on the technique used
    Many factors influence results
    Lack of standardization of the inoculums
    Thickness and quality of the culture media
    Quality and conservation of the disks
    Quality control with standardized strains
    Condition and duration of incubation
    Dr.T.V.Rao MD
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  • 35. E-test is based on arraying a concentration gradient of each antibiotic on a polymer strip. Concentration values are marked on the other side of the strip so that one can easily locate corresponding concentrations. E-strips, also known as “epsilometers”, are commercially prepared by micro dispersing robotic machines that can deliver Nano liter volumes of antibiotic concentration along the strip.
    What is e-test
    Dr.T.V.Rao MD
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  • 36. E-test
    Plastic strips with a predefined gradient of
    One antibiotic
    One antifungal
    Only one manufacturer
    One strip per antibiotic
    Wide range of antibiotics
    Easy to use
    Storage at -20°C
    Short shelf life, expensive
  • 37. An E-test combines aspects of Kirby-Bauer and MIC tests
    Figure 10.11
    Dr.T.V.Rao MD
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  • 38. Reading E-tests
    Ciprofloxacin for Yersinia pestis
    Resistant > 4 ug/ml
    Intermediate 1-4 ug/ml
    Susceptible < 1
    Upper reading
    Dr.T.V.Rao MD
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  • 39. Problems with E-test reading
    Common interpretation problems
    Dr.T.V.Rao MD
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  • 40. The Liofilchem MIC Test Strip is a quantitative assay for determining the Minimum Inhibitory Concentration (MIC) of antimicrobial agents against microorganisms to indicate appropriate patient treatment and for identifying resistance patterns
    New Liofilchem MIC Test Strips
    Dr.T.V.Rao MD
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  • 41. When the Liofilchem MIC Test Strip is applied onto an inoculated agar surface, the preformed exponential gradient of antimicrobial agent is transferred into the agar matrix. After 18 hours incubation or longer, a symmetrical inhibition ellipse centered along the strip is formed. The MIC is read directly from the scale in terms of µg/mL, at the point where the edge of the inhibition ellipse intersects with the MIC Test Strip.
    New Liofilchem MIC Test Strips
    Dr.T.V.Rao MD
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  • 42. Oxoid has l improved M.I.C.Evaluator™ (M.I.C.E.™) strips, a product range for the accurate determination of minimum inhibitory concentration (MIC) values. The new distinctive gradient format of M.I.C.E. strips provides an excellent contrast with agar and the increased font size makes reading easier.
    M.I.C.Evaluators (M.I.C.E.) Simple, Convenient Method for Accurate MIC Values
    Dr.T.V.Rao MD
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  • 43. Role of NCDC ( INDIA )in containment of Antimicrobial resistance (AMR)
    Antimicrobial resistance in pathogens causing important communicable diseases has become a matter of great public health concern globally including our country. Resistance has emerged even to newer, more potent antimicrobial agents like carbapenems. The factors responsible for this are widespread use and availability of practically all the antimicrobials across the counter meant for human, animal and industrial consumption. There are definite policies /guidelines for appropriate  use of  antimicrobials at national level in specific national health programme being run in the country
    Dr.T.V.Rao MD
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  • 44. Dr.T.V.Rao MD
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    Implementation of WHONET CAN HELP TO MONITOR RESISTANCE
    Legacy computer systems, quality improvement teams, and strategies for optimizing antibiotic use have the potential to stabilize resistance and reduce costs by encouraging heterogeneous prescribing patterns and use of local susceptibility patterns to inform empiric treatment.
  • 45. Antibiotic Resistance a concern to humanity ???
    Dr.T.V.Rao MD
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  • 46. Dr.T.V.Rao MD
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    Follow me for Articles of Interest on Microbiology ..
  • 47. Created by Dr.T.V.Rao MD for ‘ e ‘ learning resources for Medical Microbiologists in the Developing World
    Email
    doctortvrao@gmail.com
    Dr.T.V.Rao MD
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